Irreversible electroporation using nanoparticles

ABSTRACT

The present invention provides methods, devices, and systems for in vivo treatment of cell proliferative disorders. The invention can be used to treat solid tumors, such as brain tumors. The methods rely on non-thermal irreversible electroporation (IRE) to cause cell death in treated tumors. In embodiments, the methods comprise the use of high aspect ratio nanoparticles with or without modified surface chemistry.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation-In-Part application of U.S.patent application Ser. No. 12/491,151, filed on 24 Jun. 2009, theentire disclosure of which is hereby incorporated herein by reference,which application is a continuation-in-part application of U.S. patentapplication Ser. No. 12,432,295, filed on Apr. 29, 2009, whichapplication claims priority to U.S. provisional application Ser. No.61/125,840, filed on Apr. 29, 2008; U.S. patent application Ser. No.12/491,151 also claims the benefit of the filing date of U.S.provisional application, Ser. No. 61/171,564, filed on Apr. 22, 2009,U.S. provisional application Ser. No. 61/167,997, filed on Apr. 9, 2009,and U.S. provisional application Ser. No. 61/075,216, filed on Jun. 24,2008. Applicants claim benefit of the filing date of U.S. patentapplication Ser. No. 12/491,151 and U.S. provisional application Ser.No. 61/167,997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of biomedical engineering andmedical treatment of diseases and disorders. More specifically, theinvention relates to devices and methods for destroying aberrant cellmasses, including tumor tissues, such as cancerous tissues of the brainand leukemias.

2. Description of Related Art

Treatment of abnormal cell growth in or on normal body tissues andorgans can be achieved in many different ways to achieve reduced cellgrowth, reduction of the resulting aberrant cell mass, and evendestruction of the aberrant cell mass. In general, treatments known inthe art involve surgical intervention to physically remove the aberrantcell mass, radiation to kill the cells of the aberrant cell mass,exposure of aberrant cells to toxic chemicals (i.e., chemotherapy), or acombination of two or all three of these. While each treatment modalityhas shown significant effectiveness in treatment of various cellproliferative diseases, no one technique has been shown to be highlyeffective at treating all types of cell proliferative diseases anddisorders. Furthermore, each technique has significant drawbacks. Forexample, surgical intervention is highly effective at removal of solidtumors on tissues and organs that are physically accessible and capableof sustaining physical damage or capable of regeneration. However,surgical intervention can be difficult to perform on tumors that are notreadily accessible or on organs that do not regenerate (e.g., braintumors), and can involve substantial physical damage to the patient,requiring extensive recuperation times and follow-on treatments.Likewise, treatment with radiation can result in collateral damage totissue surrounding the tumor, and can cause long-lasting side-effects,which can lower the quality of life of the patient. Similarly,chemotherapeutic treatments cause systemic damage to the patient, andcan result in significant side-effects that might require a longrecuperation period or permanent damage to the patient.

In the treatment of tumors, including malignant tumors, it is recognizedin the medical arts that it is important to achieve ablation of theundesirable tissue in a well-controlled and precise way withoutaffecting the surrounding healthy tissue. The inventors and theircolleagues recently developed a new method to treat tumors, known asirreversible electroporation (IRE). The procedure involves placingelectrodes within or near the targeted region to deliver a series of lowenergy, microsecond electric pulses for approximately 1 minute. Thesepulses permanently destabilize the cell membranes of the targeted tissue(e.g., tumor), thereby killing the cells. IRE does not affect majorblood vessels, does not require the use of drugs and non-thermally killsneoplastic cells in a precise and controllable manner, withoutsignificantly damaging surrounding tissue. The inventors and theircolleagues also recently showed the complete regression in 12 out of 13treated tumors in vivo using IRE on a type of aggressive sarcomaimplanted in nude mice (Al-Sakere, B. et al., 2007, “Tumor ablation withirreversible electroporation.” PLoS ONE 2.).

Although advances have been made recently in the field of IRE and theconcept of treatment of tumors with IRE has been established, thepresent inventors have recognized that there still exists a need in theart for improved devices and methods for ablating diseased or disorderedtissues, such as tumor tissues, using IRE. The present inventionaddresses those needs.

SUMMARY OF THE INVENTION

The present invention provides an advancement over tissue ablationtechniques previously devised by providing improved devices and methodsfor precisely and rapidly ablating diseased, damaged, disordered, orotherwise undesirable biological tissues in situ. As used herein, theterm ablation is used to indicate destruction of cells, but notnecessarily destruction of the underlying extracellular matrix. Morespecifically, the present invention provides new devices and methods forablating target tissues for the treatment of diseases and disorders, andparticularly neoplasias, including both solid tumors and leukemias,using IRE in combination with nanoparticles. Use of IRE to decellularizediseased tissue provides a controlled, precise way to destroy aberrantcells of a tissue or organ, such as tumor or cancer cells or masses.

Non-thermal IRE is a method to kill undesirable cells using electricfields in tissue while preserving the ECM, blood vessels, and neuraltubes/myelin sheaths. Certain electrical fields, when applied across acell, have the ability to permeabilize the cell membrane through aprocess that has come to be called “electroporation”. When electricalfields permeabilize the cell membrane temporarily, after which the cellssurvive, the process is known as “reversible electroporation”.Reversible electroporation has become an important tool in biotechnologyand medicine. Other electrical fields can cause the cell membrane tobecome permeabilized, after which the cells die. This deadly process isknown as “irreversible electroporation”. According to the presentinvention, non-thermal irreversible electroporation is a minimallyinvasive surgical technique to ablate undesirable tissue, for example,neoplastic cells or tissues. The technique is easy to apply, can bemonitored and controlled, is not affected by local blood flow, and doesnot require the use of adjuvant drugs. The minimally invasive procedureinvolves placing needle-like electrodes into or around the targeted areato deliver a series of short and intense electric pulses that inducestructural changes in the cell membranes that promote cell death. Thevoltages are applied in order to electroporate tissue without inducingsignificant Joule heating that would significantly damage major bloodvessels and the ECM. For a specific tissue type and set of pulseconditions, the primary parameter determining the volume irreversiblyelectroporated is the electric field distribution within the tissue.Recent IRE animal experiments have verified the many beneficial effectsresulting from this special mode of non-thermal cell ablation, such aspreservation of major structures including the extracellular matrix,major blood vessels, and myelin sheaths, no scar formation, as well asits promotion of a beneficial immune response.

In a first aspect, the present invention provides a method for treatingaberrant cell growth in animals. In general, the method comprisesinserting one or more electrodes into or immediately adjacent toaberrant cell masses or in a region of neoplastic cell growth andapplying IRE to cause irreversible cell death to the aberrant cells. Insome embodiments, two or more electrodes are used to treat aberrantcells and effect cell death. The electrodes may be present on the sameor different devices. Preferably, the parameters for IRE are selected tominimize or avoid excessive heating of the treated tissue andsurrounding tissue, thus reducing collateral damage to healthy tissuenear the aberrant cells being treated. In addition, it is preferable tominimize the total electrical charge delivered when treating tissues,such as brain tissue, to avoid complications. The methods can be appliedto treat a number of neoplasias, including any number of solid tumors,such as liver cancer, prostate cancer, and pancreatic adenocarcinoma.Exemplary in vivo therapeutic methods include regeneration of organsafter treatment for a tumor, preparation of a surgical site forimplantation of a medical device, skin grafting, and replacement of partor all of a tissue or organ, such as one damaged or destroyed by adisease or disorder (e.g., the liver). Exemplary organs or tissuesinclude: heart, lung, liver, kidney, urinary bladder, brain, ear, eye,or skin. In view of the fact that a subject may be a human or animal,the present invention has both medical and veterinary applications.

Viewed differently, the method for treating aberrant cell growth inanimals can be considered a method of treating an animal (includinghumans) having an aberrant cell growth or mass in or on a tissue or anorgan. In exemplary embodiments, the organ is a brain, and the aberrantcell mass is a benign or malignant tumor. In other exemplaryembodiments, the cells to be treated are leukemia cells, such as thoseresident in bone marrow. Under this view, the method can be a method oftreating an animal suffering from a disease or disorder resulting fromaberrant cell growth by reducing or eliminating some or all of a mass(e.g., tumor) produced by the aberrant cell growth. In certainembodiments, the aberrant cell mass to be treated includes cells withcell cycling dysfunctions, including cells that are not dying orundergoing apoptotic mechanisms of cell death or similar programmed celldeath at appropriate, natural, or naturally induced times, such as thosemediated through protein bindings or intracellular cascades. Inaddition, the aberrant cell mass in certain embodiments includes cellswith alterations (including genetic and protein expression andpost-translational alterations and modifications) leading to immunesystem evasion or immune system indifference.

In embodiments of the methods of the present invention, IRE withnanoparticles is used to increase the treatment area without increasingthe applied voltage and without causing significant thermal damage.Nanoparticles offer a promising solution for treatment of neoplasiasbecause of their size (about 1 to about 1,000 nm) and ability to diffusethrough extracellular spaces. The three most important properties of ananoparticle for enhancing electric fields are its shape, orientationwith respect to the applied field, and electrical properties(conductivity and permittivity). The contribution of each of theseproperties towards enhancing electric fields are shown in FIG. 18 for aspherical nanoparticle, a rod-shaped particle oriented perpendicular tothe applied field, and a rod-shaped particle oriented parallel to theapplied field with a range of conductivity and permittivity ratios.However, nanoparticle embodiments suitable for IRE are not limited torod or spherical shaped particles. Other nanoparticle embodiments mayinclude, but are not limited to, fullerenes (a.k.a. C₆₀, C₇₀, C₇₆, C₈₀,C₈₄), endohedral metallofullerenes (EMFs) (a.k.a. buckyballs, whichcontain additional atoms, ions, or clusters inside their fullerenecage), trimetallic nitride templated endohedral metallofullerenes (TNTEMFs, high-symmetry four-atom molecular cluster endohedrals which areformed in a trimetallic nitride template within the carbon cage),single-walled and multi-walled carbon nanotubes, branched and dendriticcarbon nantotubes, gold nanorods, silver nanorods, single-walled andmulti-walled boron/nitrade nanotubes, carbon nanotube peapods (nanotubeswith internal metallo-fullerenes and/or other internal chemicalstructures), carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles,nanorods, and cellulose nanoparticles. The particle embodiment can alsoinclude microparticles with the capability to enhance IRE effectivenessor selectivity. Other non-limiting exemplary nanoparticles include glassand polymer micro- and nano-spheres, biodegradable PLGA micro- andnano-spheres, gold, silver, carbon, and iron nanoparticles. Inembodiments, the nanoparticles of the methods of the invention,including insulative and conductive nanoparticles of varying shape, canenhance pulsed electric field therapies by lowering the electric fieldthreshold required for inducing IRE and enlarging the treatable area.

The nanoparticles can comprise modified surface chemistries to cause thelocalized destruction of targeted cells, while leaving untargeted cellsintact. Although IRE has been shown to promote tumor regression, itcannot selectively kill cancer cells within a tumor mass without alsokilling healthy cells. The selectivity of pulsed electric fieldtherapies can be enhanced through the use of nanoparticles that can befunctionalized to target specific cancer cells using various antibodiesor chemical compounds. These methods can be employed to reduce oreliminate neoplastic cells from a subject within and beyond thetreatment margin, while maintaining proper organ function.

To effect the methods according to the invention, the present inventionprovides devices designed to treat aberrant cell masses usingirreversible electroporation (IRE). While IRE devices have beendisclosed prior to the priority date of this document, advanced surgicaltools for in vivo IRE to treat diseased tissues and organs had not beendeveloped. The present invention provides devices suitable for in vivoIRE treatment of diseases and disorders, particularly those associatedwith abnormal cell growth in or on a tissue or organ, which allow forminimally invasive treatment of patients suffering from such abnormalcell growth. The present inventors have designed microsurgical tools totreat currently inoperable tumors in humans and other animals throughIRE. While not so limited, the designs provided herein are sufficient toablate the majority of tumors smaller than about 3 cm in diameter, suchas those about 14 cc in volume or less. The devices and methods of theinvention are also suitable for treatment of non-solid tumors, such asleukemias.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention, and together with the written description, serve to explaincertain principles of the invention.

FIGS. 1A-1C show magnetic resonance imaging (MRI) images of tissue afternon-thermal IRE on canine tissue. The images show that non-thermal IREdecellularization zones were sharply demarcated T1 iso- to hypo-intense,T2 hyperintense and mild and peripherally contrast enhancing followingintravenous administration of gadolinium, consistent with fluidaccumulation within decellularization sites and a focal disruption ofthe blood-brain-barrier. FIG. 1A shows an MRI before IRE, T2 weighted;FIG. 1B shows superficial non-thermal IRE decellularization site, T2weighted; and FIG. 1C shows post-contrast T1 weighted; the dog's rightis conventionally projected on the left.

FIG. 2 shows an ultrasound image of brain tissue 24 hour post-IREtreatment. The IRE decelluarization zone is clearly visible as a welldemarcated, hypoechoic circular lesion with a hyperechoic rim.

FIG. 3 depicts images of brain tissue after non-thermal IRE treatment.

FIG. 4A shows a sharp delineation of brain tissue showing the regions ofnormal and necrotic canine brain tissue after IRE. FIG. 4B shows IREtreated brain tissue showing sparing of major blood vessels.

FIG. 5 shows a three-dimensional MRI source reconstruction of asuperficial lesion site.

FIG. 6 shows a bar graph indicating results of IRE performed in vitro onJ3T glioma cells at different pulse values.

FIGS. 7A-7E depict various exemplary embodiments of a device accordingto the invention. FIG. 7A depicts a device, showing a connector, wiring,and electrodes disposed at the tip. FIGS. 7B-7E depict alternativeplacement of electrodes, which can be retractable.

FIGS. 8A-8C depict an expanded view of an electrode tip according to oneembodiment of the invention. FIG. 8A depicts an exploded view of thevarious concentric layers of materials making up the electrode tip. FIG.8B depicts a side view of the electrode of FIG. 8A, showing the variouslayers in cut-away fashion. FIG. 8C depicts the electrode tip viewedalong the proximal-distal plane.

FIGS. 9A and 9B depict an embodiment of an assembled electrode tip foran exemplary treatment where the tip is inserted within a tumor embeddedwithin benign tissue. FIGS. 9A and 9B depict an embodiment of the deviceof the invention, comprising a hollow core for delivery of bioactiveagents.

FIG. 10 depicts yet another embodiment of a device according to theinvention, in which the outer, non-conductive sheath is adjustable toallow for selection of an appropriate depth/length of electricallyconductive material to be exposed to tissue to be treated. Theembodiment includes screw tappings (not shown) to allow real-timeadjustment of the electrode layer lengths to customize electrodedimensions prior to a procedure.

FIG. 11 depicts an exemplary system according to the invention, whichincludes an adjustable height electrode, a handle for physician guidanceof the device into the patient, and a power source/controller to provideand control electrical pulses.

FIGS. 12A-12E depict electrical field outputs from variousconfigurations employing two electrodes. FIG. 12A depicts the use of twoelectrodes spaced 0.5 cm apart. FIG. 12B depicts the use of twoelectrodes spaced 1.0 cm apart. FIG. 12C depicts the use of twoelectrodes spaced 1.5 cm apart. FIG. 12D depicts the use of twoelectrodes spaced 1.0 cm apart. FIG. 12E depicts a side view ofelectrical field outputs from one device having two electricallyconductive regions separated by an insulative region of a length of 0.5cm.

FIGS. 13A-13C depict electrical field outputs from variousconfigurations employing three needle electrodes having differentdiameters. FIG. 13A depicts the use of electrodes of 2 mm, 0.5 mm, and 1mm (from left to right). FIG. 13B depicts the use of electrodes of 2 mm,1 mm, and 0.5 mm (from left to right). FIG. 13C depicts the use ofelectrodes of 1 mm, 2 mm, and 0.5 mm (from left to right).

FIGS. 14A-14J depict electrical field outputs for various combinationsof electrodes emitting different charges. FIG. 14A depicts atwo-dimensional display for the use of four electrodes of alternatingpolarity. FIG. 14B depicts an axis symmetric display for the use of foursimilar electrodes of alternating polarity. FIG. 14C depicts atwo-dimensional display for the use of four charged electrodes, thecenter two at 5000V and 0V and the outer two at 2500V. FIG. 14D depictsan axis symmetric display for the use of a similar electrode set up asFIG. 14C. FIG. 14E depicts a two-dimensional display for the use ofthree electrodes with the center one at 2500V and the outer two at 0V.FIG. 14F depicts an axis symmetric display for the use of threeelectrodes similar to FIG. 14E. FIG. 14G depicts a two-dimensionaldisplay for the use of three charged electrodes, the center at 0V, theleft at 5000V, and the right at 2500V. FIG. 14H depicts an axissymmetric display for the use of a similar electrode set up as FIG. 14G.FIG. 14I depicts a two-dimensional display for the use of three chargedelectrodes, the center at 1750V, the left at 3000V, and the right at 0V.FIG. 14J depicts an axis symmetric display for the use of a similarelectrode set up as FIG. 14I.

FIGS. 15A-15D depict thermal effects from use of three needleelectrodes, with and without use of a cooling element in the electrode.FIG. 15A shows thermal effects without cooling, while FIG. 15B shows thethermal effects under the same pulsing conditions, but with electrodecooling. FIG. 15C shows thermal effects without cooling, while FIG. 15Dshows the thermal effects under the same pulsing conditions, but withelectrode cooling.

FIGS. 16A-16C depict thermal effects from use of two bipolar electrodesand an intervening balloon.

FIG. 17 depicts a gold nanorod (Au NR) with modified surface chemistrythat can be used to selectively target and kill a cancer cell whenplaced in an electric field.

FIG. 18 depicts the results of a finite-element model showing the IREtreatment area (electric field >600 V/cm) normalized by the area of thenanoparticle for varying ratios of conductivity (σ) and permittivity (ε)between the nanoparticle (p) and surrounding medium (m) when thenanoparticles are placed in a uniform electric field of 500 V/cm. Theresults were obtained by solving the complex Laplace equation in a FEMwith an adaptive mesh and performing a subdomain integration within thenanoparticle and its surrounding medium.

FIG. 19 depicts a bar graph showing the effect of electric fieldstrength on cell viability following IRE treatment with and withoutnanoparticles.

FIG. 20 depicts the results of a finite-element model showing the effectof polystyrene particles on delivery of electrical energy to a cell. Acell (14 μm diameter) with and without surrounding polystyrene beads (1μm diameter) in a 1000 V/cm uniform DC electric field (upper left image)and a 1 MHz AC electric field (lower left image). The surface color maprepresents the electric field (V/cm), and the contours represent theelectric potential (V). The induced transmembrane voltage (ITV) tracedclockwise around a cell (14 μm diameter) with (wavy line trace) andwithout (smooth line trace) surrounding polystyrene beads (1 μmdiameter) in a 1000 V/cm uniform DC electric field (upper right graph)and a 1 MHz AC electric field (lower right graph).

FIG. 21 depicts the results of a finite-element model for predicting thetreatment area resulting from an IRE procedure using 2 needleelectrodes. The leftmost graph of FIG. 21 shows the resulting electricfield distribution (contour) and temperature rise (surface). Therightmost image of FIG. 21 shows a ratio of the electric field withinthe tissue to the electric field threshold for IRE (500 V/cm asdetermined experimentally) versus the area encompassed by the electricfield within the tissue.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

Reference will now be made in detail to various exemplary embodiments ofthe invention. It is to be understood that the following discussion ofexemplary embodiments is not intended as a limitation on the invention,as broadly disclosed above. Rather, the following discussion is providedto give the reader a more detailed understanding of certain aspects andfeatures of the invention.

Before embodiments of the present invention are described in detail, itis to be understood that the terminology used herein is for the purposeof describing particular embodiments only, and is not intended to belimiting. Further, where a range of values is provided, it is understoodthat each intervening value, to the tenth of the unit of the lowerlimit, unless the context clearly dictates otherwise, between the upperand lower limits of that range is also specifically disclosed. Eachsmaller range between any stated value or intervening value in a statedrange and any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included or excluded in the range,and each range where either, neither, or both limits are included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a pulse” includes aplurality of such pulses and reference to “the sample” includesreference to one or more samples and equivalents thereof known to thoseskilled in the art, and so forth. Furthermore, the use of terms that canbe described using equivalent terms include the use of those equivalentterms. Thus, for example, the use of the term “patient” is to beunderstood to include the terms “subject”, “animal”, “human”, and otherterms used in the art to indicate one who is subject to a medicaltreatment.

Electroporation is the phenomenon in which permeability of the cellmembrane to ions and macromolecules is increased by exposing the cell toshort (microsecond to millisecond) high voltage electric pulses. Theapplication of the electric pulses can have no effect, can have atransient effect known as reversible electroporation, or can causepermanent permeation known as irreversible electroporation (IRE), whichleads to non-thermal cell death by necrosis.

Davalos, Mir, and Rubinsky (Davalos, R. V. et al., 2005, “Tissueablation with irreversible electroporation.” Annals of BiomedicalEngineering, 3(2):223-231) recently postulated and demonstrated that IREcan be used as an independent drug-free tissue ablation modality forparticular use in cancer therapy. This minimally invasive procedureinvolves placing electrodes into or around the targeted area to delivera series of short and intense electric pulses to induce the irreversiblestructural changes in the membranes. To achieve IRE, the electric fieldin the targeted region needs to be above a critical value, which isdependent on a variety of conditions such as tissue type and pulseparameters.

The present invention extends and improves on prior techniques for IREby providing new methods and devices for IRE treatment of neoplasias,including those associated with brain cancer. Because the brain issusceptible to small fluctuations in temperature, the present inventionprovides devices and techniques for non-thermal IRE to kill undesirablecells and tissues. In addition, because the brain functions by way ofelectrical charges, the present invention provides devices andtechniques that limit or precisely control the amount of electricalcharge delivered to tissue. Other neoplasias also show similarsensitivities, and the invention is equally applicable to allneoplasias. To achieve the invention, a device has been developed thatcontains both conducting and non-conducting surfaces and that is capableof delivering controlled pulses of electricity to tumor tissues whilesubstantially protecting surrounding healthy tissue. In exemplaryembodiments, the device has a laminate structure of at least oneelectrically conductive and at least one electrically insulativematerial. In some exemplary embodiments, the device has at least twoconcentric disk electrodes separated by an insulating material similarin dimensions to those already used in deep brain stimulation (DBS). DBSis an FDA approved therapy that alleviates the symptoms of otherwisetreatment-resistant disorders, such as chronic pain, Parkinson'sdisease, tremor, and dystonia. The Examples, below, present resultsdemonstrating that an IRE procedure does not induce substantial thermaleffects in the brain and bone marrow, and delivers electrical charges tohighly defined regions of tissues, supporting the conclusion that IREcan be used as a minimally invasive surgical technique for the treatmentof brain cancer and other diseases and disorders involving aberrant cellmass development. The methods employ the unique designs discussedherein, which provide improved controlled delivery of electrical pulseswith controlled three-dimensional patterns and controlled thermaloutputs. The present devices and systems provide surgical tools andmethods for IRE treatment of subcutaneous tumors that expand theapplication space for this new technology, with the potential to treat anumber of solid tumor cancers, including brain, liver, prostate andpancreatic adenocarcinoma and non-solid tumor cancers, including alltypes of leukemia.

The following detailed description focuses mainly on devices, systems,and methods for treatment of brain cancer. However, those of skill inthe art will recognize that the concepts discussed have equivalentapplication to other diseases and disorders involving aberrant cellgrowth and/or production of deleterious cell masses on or by organs andtissues.

While the prognosis for many patients has improved with new drugs andradiosurgery, options to treat primary brain tumor patients are limitedbecause of the need for techniques to be non-thermal, i.e., notpropagate a convective hot spot in normal brain tissue not beingtreated. The current invention allows for IRE as an extremely useful,minimally invasive surgical technique for the treatment of brain cancerand other neoplasias. The present designs for a surgical tool/treatmentsystem for brain cancer is readily translated into the development oftools for other types of cancer.

As mentioned above, the present invention provides a method for treatingaberrant cell growth in animals. The aberrant cell growth can be anytype of aberrant cell growth, but in exemplary embodiments detailedherein, it is generally described in terms of tumors, such as braintumors. In general, the method of treating comprises temporarilyimplanting one or more electrodes, which may be present on the same ordifferent devices, into or immediately adjacent a neoplasia, andapplying an electrical field to the neoplasia in multiple pulses orbursts over a prescribed or predetermined period of time to causeirreversible cell death to some or all of the neoplastic cells.Preferably, irreversible damage to non-neoplastic cells in proximity tothe treated neoplastic cells is minimal and does not result insignificant or long-lasting damage to healthy tissues or organs (or asignificant number of cells of those tissues or organs). According tothe method of the invention, cell killing is predominantly, essentially,or completely due to non-thermal effects of the electrical pulsing. Themethod further comprises removing the electrode(s) after suitabletreatment with the electrical fields. In preferred embodiments, themethod also includes administering to the subject being treated aneffective amount of a nanoparticle such that the nanoparticlesinfiltrate the neoplastic cell mass that is to be treated with IRE. Thepresence of the nanoparticles at the site of treatment improvestreatment outcome. As a general matter, because the method involvestemporary implantation of relatively small electrodes, it is minimallyinvasive and does not result in the need for significant post-treatmentprocedures or care. Likewise, it does not result in significantancillary or collateral damage to the subject being treated.

In practicing the method, the number of electrodes, either on a singleor multiple devices, used can be selected by the practitioner based onthe size and shape of the neoplasia to be treated and the size and shapeof the electrode. Thus, embodiments of the invention include the use ofone, two, three, four, five, or more electrodes. Each electrode can beindependently sized, shaped, and positioned in or adjacent the tissue tobe treated. In addition, the number and spacing of electrodes on asingle device can be adjusted as desired. As detailed below, thelocation, shape, and size of electrodes can be selected to producethree-dimensional killing zones of numerous shapes and sizes, allowingfor non-thermal treatment of neoplasias of varying shapes and sizes.

Surprisingly, it has been found that pulse durations for ablation ofneoplasias, and in particular solid tumors, can be relatively short,thus reducing the probability of generation of thermal conditions andexcessive charges that cause collateral damage to healthy tissues. Morespecifically, the present invention recognizes for the first time that,in contrast to prior disclosures relating to IRE, the pulse length forhighly efficient tissue ablation can be lower than 100 microseconds (100μs). Indeed, it has surprisingly been determined that a pulse length of25 microseconds (25 μs) or lower can successfully cause non-thermal celldeath. Thus, in embodiments, the method of treatment uses pulse lengthsof 10 μs, 15 μs, 20 μs, 25 μs, 30 μs, 35 μs, 40 μs, 45 μs, 50 μs, 55 μs,60 μs, 65 μs, 70 μs, 75 μs, 80 μs, 85 μs, or 90 μs. Preferably, to mosteffectively minimize peripheral damage due to heat, pulse lengths arelimited to 90 μs or less, for example 50 μs or less, such as 25 μs. Byreducing the pulse length, as compared to prior art techniques for IRE,larger electric fields can be applied to the treatment area whileavoiding thermal damage to non-target tissue (as well as to targettissue). As a result of the decreased pulse length and concomitantreduction in heat production, the methods of the invention allow fortreatment of tissues having higher volumes (e.g., larger tumors,non-solid tumors) than possible if prior art methods were to be employedfor in vivo treatment of tumors.

It has also been determined that voltages traditionally used for IRE aretoo high for beneficial treatment of tumors in situ. For example,typically, IRE is performed using voltages of between 4000 V/cm to 1500V/cm. The present invention provides for use of voltages of much lowerpower. For example, the present methods can be performed using less than1500 V/cm. Experiments performed by the inventors have shown that 2000V/cm can cause excessive edema and stroke in patients when applied tobrain tissue. Advantageously, for treatment of brain tumors, appliedfields of about 500 V/cm to 1000 V/cm are used. Thus, in general fortreatment of brain tumors, applied fields of less than 1000 V/cm can beused.

Further, it has been discovered that the number of electrical pulsesthat can be applied to successfully treat tumors can be quite high.Prior art methods of using IRE for various purposes included the use ofrelatively few pulses, for example 8 pulses or so. Reports of use of upto 80 pulses for IRE have been published; however, to the inventors'knowledge, a higher number of pulses has not been recommended. Thepresent invention provides for the use of a relatively high number ofpulses, on the order of 90 pulses or greater. For example, in exemplaryembodiments, 90 pulses are used. Other embodiments include the use ofmore than 90 pulses, such as 100 pulses, 110 pulses, or more. Moderatelyhigh numbers of pulses, such as those in the range of 50-90 pulses canalso be used according to the invention, as well as moderate numbers ofpulses, such as those in the range of 20-50 pulses.

According to the method of the invention, cycle times for pulses are setgenerally about 1 Hz. Furthermore, it has been found that alternatingpolarity of adjacent electrodes minimizes charge build up and provides amore uniform treatment zone. More specifically, in experiments performedby the inventors, a superficial focal ablative IRE lesion was created inthe cranial aspect of the temporal lobe (ectosylvian gyrus) using theNanoKnife®B (Angiodynamics, Queensbury, N.Y.) generator, blunt tipbipolar electrode (Angiodynamics, No. 204002XX) by delivering 9 sets often 50 μs pulses (voltage-to-distance ratio 2000 V/cm) with alternatingpolarity between the sets to prevent charge build-up on the stainlesssteel electrode surfaces. These parameters were determined from ex-vivoexperiments on canine brain and they ensured that the charge deliveredduring the procedure was lower than the charge delivered to the humanbrain during electroconvulsive therapy (an FDA approved treatment formajor depression). Excessive charge delivery to the brain can inducememory loss, and thus is preferably avoided.

The method of the invention encompasses the use of multiple electrodesand different voltages applied for each electrode to precisely controlthe three-dimensional shape of the electric field for tissue ablation.More specifically, it has been found that varying the amount ofelectrical energy emitted by different electrodes placed in a tissue tobe treated allows the practitioner to finely tune the three-dimensionalshape of the electrical field that irreversibly disrupts cell membranes,causing cell death. Likewise, the polarity of electrodes can be variedto achieve different three-dimensional electrical fields. Furthermore,one of the advantages of embodiments of the invention is to generateelectric field distributions that match complex tumor shapes bymanipulating the potentials of multiple electrodes. In theseembodiments, multiple electrodes are energized with different potentialcombinations, as opposed to an “on/off” system like radio frequencyablation, to maximize tumor treatment and minimize damage to surroundinghealthy tissue.

According to the method of the invention, the separation of theelectrodes within or about the tissue to be treated can be varied toprovide a desired result. For example, the distance between two or moreelectrodes can be varied to achieve different three-dimensionalelectrical fields for irreversible disruption of cell membranes. Thethree-dimensional shape can thus be set to ablate diseased tissue, butpartially or completely avoid healthy tissue in situations where theinterface between healthy and diseased tissue shows a complex threedimensional shape.

The methods of the invention are well suited for treatment of solidtumors and other neoplasias using non-thermal IRE. To better ensure thatcell ablation is a result of non-thermal effect, and to better protecthealthy tissue surrounding the site of treatment, the method can furthercomprise cooling the electrodes during the treatment process. Byapplying a heat sink, such as a cooling element in an electrode(discussed below), generation of heat in and around tissue in closeproximity to the electrodes can be minimized, resulting in a moreconsistent application of non-thermal IRE to the tissue and a morecontrolled application of cell killing to only those tissues desired tobe treated.

The method of the invention, in embodiments, includes the use ofelectrodes of different sizes and shapes. Studies performed by theinventors have shown that the electrical field distribution may bealtered by use of electrodes having different diameters, lengths, andshapes. Thus, the use of different sizes and shapes of conductingsurfaces can be used to control the electrical fields used for cellablation. In certain embodiments, the method includes the use of avariable size electrode. For example, an electrode may be used that, inone configuration has a relatively small diameter, which is used forminimally invasive implantation of the electrode into the site to betreated. Once inserted, a sheath or other covering can be retracted toallow expansion of the electrode tip to a different size for applicationof the electric field. After treatment, the sheath can be moved to coverthe tip again, thus reducing the size of the tip to its original size,and the electrode withdrawn from the treated tissue. The expandableelement can be thought of as a balloon structure, which can have varyingdiameters and shapes, depending on original material shape and size.

In embodiments, the method of the invention comprises the use ofnanoparticles, as shown, for example, in FIG. 18. In the presence of anelectric field, a nanoparticle, such as a nanosphere or nanorod,experiences a charge polarization. In the case of a conductive nanorod,this polarization is larger in the direction parallel to the axis thanthe radial direction, and charge accumulates at the tips, forming adipole. The dipole serves to simultaneously align the nanoparticle inthe direction of the electric field and locally amplify the electricfield at the nanoparticle tips. In this scenario, the nanorod behaveslike a downscaled dipole antenna from classical antenna theory, whichindicates that a higher aspect ratio nanoparticle is more effective atconcentrating the field. Conversely, if an insulative nanorod isemployed, a large field enhancement can still be obtained if the longdimension of the particle is oriented perpendicular to the appliedelectric field, and a higher aspect ratio particle still serves toenhance this effect. Field enhancement can also be obtained for aconductive and insulative sphere, while to a lesser extent than thescenarios described above. However, when multiple spheres are employed,the high surface charge density concentrates the electric field betweenthe spheres, as shown, for example, in FIG. 20. In other words, thefield enhancement of closely spaced spheres becomes very strong, and ison the order of that seen in prolate nanoparticles. Examples ofnanoparticles include, but are not limited to, fullerenes (a.k.a. C₆₀,C₇₀, C₇₆, C₈₀, C₈₄), endohedral metallofullerenes (EMFs) (a.k.a.buckyballs, which contain additional atoms, ions, or clusters insidetheir fullerene cage), trimetallic nitride templated endohedralmetallofullerenes (TNT EMFs, high-symmetry four-atom molecular clusterendohedrals, which are formed in a trimetallic nitride template withinthe carbon cage), single-walled and multi-walled carbon nanotubes,branched and dendritic carbon nantotubes, gold nanorods, silvernanorods, single-walled and multi-walled boron/nitrade nanotubes, carbonnanotube peapods (nanotubes with internal metallo-fullerenes and/orother internal chemical structures), carbon nanohorns, carbon nanohornpeapods, liposomes, nanoshells, dendrimers, quantum dots,superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles.The particle embodiment can also include microparticles with thecapability to enhance IRE effectiveness or selectivity. Othernon-limiting exemplary nanoparticles include glass and polymer micro-and nano-spheres, biodegradable PLGA micro- and nano-spheres, gold,silver, carbon, and iron nanoparticles.

As should be evident by the present disclosure, the invention alsoencompasses use of particles on the micrometer scale in addition to thenanometer scale. Where microparticles are used, it is preferred thatthey are relatively small, on the order of 1-50 micrometers. For ease ofdiscussion, the use herein of “nanoparticles” encompasses truenanoparticles (sizes of from about 1 nm to about 1000 nm),microparticles (e.g., from about 1 micrometer to about 50 micrometers),or both. In preferred embodiments, the nanoparticles do not comprisecarbon and/or are not carbon nanotubes.

Nanoparticles, in embodiments, may be modified with surface chemistry tocause the localized destruction of targeted cells, while leavinguntargeted cells intact. The surface of nanoparticles can befunctionalized to target specific cancer cells with various antibodiesand chemical compounds. Due to the electric field enhancement and theability of functionalized nanoparticles to target cancer cells, electricpulse protocols can be optimized, such that only cancer cells withselectively bound nanoparticles experience a localized electric fieldabove the threshold for achieving IRE, and healthy cells remain intact.This methodology can be employed to purge a subject of cancer cellswithin and beyond the treatment margin, while maintaining proper organfunction. Tumors can be comprised of as much as 80% healthy cells, andselectively destroying the cancer cells, including cancer stem cells(CSCs), reduces the potential for tumor recurrence.

There are many examples of nanoparticle targeting techniques known inthe art. For example, folic acid conjugations can be used to selectivelybind to cancer cells with up-regulated folate receptors, such as breastand brain cancer cells (see FIG. 17). As another example, antibodies canbe conjugated to selectively bind to cancer cells presenting distinctantigens, such as leukemic cells. As a further example, the simpletendency of well suspended nanoparticles (coated with polymers) todiffuse into tumor masses over time following systemic delivery can beemployed as a nanoparticle targeting technique.

The methods of the invention comprise, in embodiments, treatment oftissue surrounding neoplastic tissue, particularly in the context ofsolid tumors. The surrounding tissue is treated by way of reversibleelectroporation. As such, bioactive agents can be introduced into thereversibly electroporated cells. In such embodiments, additional cellkilling, under controlled conditions, can be effected in healthy tissue.Such a treatment is preferred when treating highly aggressive malignanttumors, which often show invasion of healthy tissue surrounding thetumor. Alternatively, the bioactive agents can provide a protectiveeffect to cells in which they are introduced via reversibleelectroporation.

In embodiments, the method for treating aberrant cell growth in animalsis a method of treating a subject suffering from a neoplasia. It thusmay be a method of treating a subject suffering from cancer. Usingdifferent terminology, the method can be a method of treating aneoplasia or a method of treating cancer. As such, the method can be amethod of treating either a benign tumor or a malignant tumor. Inembodiments, the method is best suited for treatment of solid tumors. Inexemplary embodiments, the method is a method of treating a subjectsuffering from a brain tumor, such as brain cancer.

In clinical settings, the method of treating according to the inventioncan have ameliorative effects or curative effects. That is, a method oftreating a subject can provide a reduction in neoplastic cell growth ofa tumor, a reduction in tumor size, or total ablation of the tumorand/or neoplastic cells.

The method of the invention can include a single round of treatment ortwo or more rounds of treatment. That is, the method of cell ablation,either intentionally or as a result of tumor size or shape, can resultin less than complete destruction of a tumor. In such a situation, themethod can be repeated one or more times to effect the desired level oftumor reduction. As the method of the invention is relatively minimallyinvasive, multiple rounds of treatment are not as harmful to the patientas multiple rounds of traditional surgical intervention.

The method of the invention can be part of a multi-modal treatment. Themethod thus may comprise other cell-killing techniques known in the art.For example, the method may further comprise exposing the neoplasticcells or tissue to radiation, or treating the patient with achemotherapeutic agent. It likewise may be performed after or betweensurgical intervention to remove all or part of a tumor. Those of skillin the art are fully aware of the parameters for treatment with othermodalities; thus, details of those treatment regimens need not bedetailed herein.

The method of the invention is implemented using devices and systems.The devices according to the invention are suitable for minimallyinvasive temporary implantation into a patient, emission of atissue-ablating level of electricity, and removal from the patient. Thedevice according to the invention thus may be used in the treatment ofneoplasias and the treatment of patients suffering from neoplasias. Thedevices can take multiple forms, based on the desired three-dimensionalshape of the electrical field for cell killing. However, in general, thedevices include two or more regions of differing conductivity. In someembodiments, the device comprises alternating regions of conductivity,for example a region of electrical conductivity, which is adjacent aregion of electrical non-conductivity, which is adjacent a differentregion of conductivity. In embodiments, the device comprises two or morelayers of conductive and insulative materials, in a laminate structurewith alternating conductive properties. To protect tissue that is not tobe treated, the outer layer can be insulative except at the region wheretreatment is to be effected. According to embodiments of the device, theamount of conductive material exposed to the tissue to be treated can beadjusted by a movable non-conductive element disposed on the outersurface of the device.

Further, in general, the device takes a rod-like shape, with onedimension (i.e., length) being substantially longer than the other(i.e., width or diameter). While exemplary embodiments are configured ina generally cylindrical shape, it is to be understood that thecross-sectional shape of the electrode can take any suitable geometricshape. It thus may be circular, square, rectangular, oval, elliptical,triangular, pentagonal, hexagonal, octagonal, etc.

The devices of the invention comprise one or more electrodes, which areelectrically conductive portions of the device. The devices are thuselectrically conductive elements suitable for temporary implantationinto living tissue that are capable of delivering an electrical pulse tothe living tissue. The device of the invention has a proximal end and adistal end. The proximal end is defined as the end at which the deviceis attached to one or more other elements, for control of the functionof the device. The distal end is defined by the end that contacts targettissue and delivers electrical pulses to the tissue. The distal end thuscomprises an exposed or exposable electrically conductive material forimplantation into a target tissue. Typically, the distal end isdescribed as including a “tip” to denote the region of the distal endfrom which an electrical pulse is delivered to a tissue. The devicefurther comprises at least one surface defining the length andcircumference of the device.

In exemplary embodiments, the device comprises a laminate structure,with alternating conductive and non-conductive or insulative layersexpanding outward from the proximal-distal center axis to the surface ofthe device. In typical embodiments, the center most layer, which showsthe smallest diameter or width, is electrically conductive and comprisesa part of the electrode tip. However, in alternative embodiments, thecenter-most layer is an open channel through which a fluid may be passedor through which additional physical elements may be placed. Yet again,the center-most layer may comprise an insulative material. Inembodiments comprising a laminate structure, the structure can provide amore customizable electric field distribution by varying the separationdistances between electrically conductive regions. Alternatively, inembodiments, certain electrically conductive regions can be exposed orconcealed by movement of an outer, non-conductive sheath. In embodimentsthat do not comprise a laminate structure, the separation lengths can beachieved by disposing on the surface non-conductive materials at variousregions.

In some embodiments, one or more substantially open channels aredisposed along the center axis or in place of one of the conductive orinsulative layers. The channel(s) may be used as heat sinks for heatproduced by the device during use. In embodiments, water or anotherfluid is held or entrained in the channel to absorb and/or remove heat.

The device of the invention comprises an electrode tip at the distalend. The electrode tip functions to deliver electrical pulses to targettissue. The tip may be represented by a single conductive layer of thedevice or may be represented by two or more conductive layers that areexposed to the tissue to be treated. Furthermore, the tip may bedesigned to have any number of geometrical shapes. Exemplary embodimentsinclude tips having a needle-like shape (i.e., electrical pulses emanatefrom a small cone-like structure at the distal end of the device) orhaving a circular shape (i.e., electrical pulses emanate from thecylindrical outer surface of the device, which is a section of thedevice where the outer insulative layer has been removed to expose thenext layer, which is conductive). For use in treatment of brain tumors,the tip advantageously comprises a blunt or rounded end to minimizelaceration of brain tissue. In embodiments, the rounded or blunt endcomprises a hole that allows for a sharp or needle-like structure to bedeployed into tumor tissue at the appropriate time.

The device comprises a proximal end, which generally functions forattachment of the device to a power source/controller and a handle. Theproximal end thus may comprise connections for electrical wires that runfrom the power source/controller to the electrically conductive layersof the device. Standard electrical connections may be used to connectthe conductive elements to the wires. In embodiments, the device isattached to a handle for ease of use by a human. While not limited inthe means for attaching the device to the handle, in embodiments, theconnection is made by way of a friction fit between the outer surface ofthe device and the handle, for example by way of an insulative O-ring(e.g., a Norprene O-ring) on the handle. In other embodiments, thedevice comprises, on its outer surface, ridges or other surface featuresthat mate with surface features present on the handle. In yet otherembodiments, the proximal end comprises one or more structures thatallow for controlled movement of the outer surface along the length ofthe device. In such embodiments, the outer surface will comprise anouter sheath that is electrically non-conductive, and which surrounds anelectrically conductive layer. Using the structures at the proximal end,the outer sheath may be moved, relative to the rest of the device, toexpose or conceal varying portions of the electrically conductivematerial beneath it. In this way, the amount of surface area of theconductive material at the tip can be adjusted to provide a desiredheight of exposure of tissue to the electrode tip. Of course, otherstructures for securely fastening the device to a holder may be used,such as clips, set screws, pins, and the like. The device is not limitedby the type of structure used to connect the device to the holder.

The device of the invention can be designed to have any desired size.Typically, it is designed to be minimally invasive yet at the same timesuitable for delivery of an effective electrical field for IRE. Thediameter or width is thus on the order of 0.5 mm to 1 cm. Preferably,the diameter or width is about 0.5 mm to about 5 mm, such as about 1 mm,2 mm, 3 mm, or 4 mm. The length of the device is not particularlylimited, but is generally set such that a surgeon can use the devicecomfortably to treat tumors at any position in the body. Thus, for humanuse, the device is typically on the order of 40 cm or less in length,such as about 30 cm, 25 cm, or 15 cm, whereas for veterinary use, thelength can be much larger, depending on the size of animal to betreated. For treatment of human brain tumors, the length can be on theorder of 40 cm.

In some embodiments, the device, or a portion of it, is flexible. Aflexible device is advantageous for use in accessing tumorsnon-invasively or minimally invasively through natural body cavities. Inembodiments where the device or a portion of it is flexible, the shapeof the device can change based on contact with body tissues, can bepre-set, or can be altered in real-time through use of wires or othercontrol elements, as known in the art, for example in use withlaparoscopic instruments.

The device of the invention can be part of a system. In addition to thedevice, the system can comprise a handle into or onto which the deviceis disposed. The handle can take any of a number of shapes, but isgenerally designed to allow a surgeon to use the device of the inventionto treat a patient in need. It thus typically has a connector forconnecting the device to the holder, and a structure for the surgeon tograsp and maneuver the device. The handle further can comprise a triggeror other mechanism that allows the surgeon to control delivery ofelectrical pulses to the device, and thus to the tissue to be treated.The trigger can be a simple on/off switch or can comprise a variablecontrol that allows for control of the amount of power to be deliveredto the device. Additionally, the handle may be created in such a mannerthat it may be attached to additional pieces of equipment, such as onesthat allow precise placement of the electrode relative to an inertial orthe patient's frame of reference, allowing steady and accurate electrodepositioning throughout an entire procedure, which may entail theapplication of electric pulses in addition to radiotherapy, imaging, andinjections (systemically and locally) of bioactive agents. Furthermore,the handle may be attached to machines that are operated remotely bypractitioners (e.g., the Da Vinci machine).

The system can further comprise a power source and/or a power controlunit. In embodiments, the power source and control unit are the sameobject. The power source provides electrical power to the device,typically by way of an electrical connection through the handle. Thepower source can be any suitable source that can deliver the properamount of electrical power to the device of the invention. Suitablepower sources are commercially available, and the invention is notlimited by the type or manufacturer. The power control unit provides theuser with the ability to set the power output and pulse time forelectrical pulses to be delivered to the device, and thus to the tissueto be treated. Suitable control units are commercially available, andthe invention is not limited by the type or manufacturer. For example,an appropriate power source/controller is available from Angiodynamics(Queensbury, N.Y.).

The device of the invention can be disposable or reusable. Where thedevice is designed to be reusable, it is preferably fabricated frommaterials that can be sterilized multiple times without destruction ofthe device. For example, the device can be fabricated fromrust-resistant metals or alloys, such as stainless steel, and plastic orother synthetic polymeric materials that can withstand cleaning andsterilization. Exemplary materials are those that can be subjected todetergents, steam heat (e.g., autoclaving), and/or irradiation for atleast one cycle of sterilization. Those of skill in the art can selectthe appropriate materials without undue experimentation, based onmaterials used in other medical devices designed to withstand commonsterilization techniques.

EXAMPLES

The invention will be further explained by the following Examples, whichare intended to be purely exemplary of the invention, and should not beconsidered as limiting the invention in any way.

As a general background to the Examples, it is noted that the inventorsand their colleagues have successfully demonstrated decellularizationusing IRE 1) in vivo and ex vivo, 2) to show that different tissues canbe utilized, 3) to show that the area affected can be predicted usingnumerical modeling, 4) to show how numerical modeling can be used toensure the ECM, blood vessels, and neural tubes are not thermallydamaged, 5) while the organ was attached to a perfusion system, 6) whiledemonstrating preservation of major vasculature and ECM, and 7) withverification through imaging.

Example 1 IRE Performance Indicia

To illustrate 1) the possibility to monitor creation of a cell-freetissue section in brain in real-time using imaging techniques, 2) thevariety of tissues that can be used, and 3) how to preserve vasculature,a healthy female purpose bred beagle was used. Nine sets of ten pulseswere delivered with alternating polarity between the sets to minimizecharge build-up on the electrode surfaces. The maximumvoltage-to-distance ratio used was 2000 V/cm because the resultingcurrent did not exceed 2 amps. The charge that was delivered to thebrain during the IRE procedure was 22.5 mC, assuming ninety pulses (50μs pulse durations) that would result from a maximum hypotheticalcurrent of 5 amps.

TABLE 1 IRE pulse parameters EXPOSURE GAP VOLTAGE TO PULSE LENGTHDISTANCE VOLTAGE DISTANCE DURATION ELECTRODES [mm] [mm] [V] RATIO [V/cm]PULSES [μs] 1 mm Monopolar 5 5 500 1000 90 50 Bipolar Standard 7 16002000 90 50

Method: After induction of general anesthesia, a routine parietotemporalcraniectomy defect was created to expose the right temporal lobe of thebrain. Two decelluarization sites were performed: 1) a deep lesionwithin the caudal aspect of the temporal lobe using a monopolarelectrode configuration (6 mm electrode insertion depth perpendicular tothe surface of the target gyrus, with 5 mm interelectrode distance), and2) a superficial lesion in the cranial aspect of the temporal lobe usinga bipolar electrode (inserted 2 cm parallel to the rostrocaudal lengthof the target gyrus, and 2 mm below the external surface of the gyrus).Intraoperative adverse effects that were encountered included grossmicrohemorrhages around the sharp monopolar electrode needles followinginsertion into the gyrus. This hemorrhage was controlled with topicalapplication of hemostatic foam. Subject motion was completelyobliterated prior to ablating the superficial site by escalating thedose of atracurium to 0.4 mg/kg. Grossly visible brain edema and surfaceblanching of the gyrus overlying the bipolar electrode decelluarizationsite was apparent within 2 minutes of completion of IRE at this site.This edema resolved completely following intravenous administration of1.0 g/kg of 20% mannitol. No adverse clinically apparent effectsattributable to the IRE procedure, or significant deterioration inneurologic disability or coma scale scores from baseline evaluationswere observed. However, the results indicated to the inventors that alower voltage would provide adequate results but with less ancillarytrauma to the brain.

Methods to monitor creation of cell-free tissues in vivo: A uniqueadvantage of IRE to ablate tissues in vivo is its ability to bemonitored in real-time using imaging techniques, such as electricalimpedance tomography, MRI, and ultrasound. Below, this Example shows MRIexaminations performed immediate post-operatively, which demonstratethat IRE decelluarization zones were sharply demarcated (FIGS. 1A-C).

As shown in FIGS. 1A-C, neurosonography performed intraoperatively andat 1 hour and 24 hours post-procedure demonstrated clearly demarcateddecellularization zones and visible needle tracts within the targetedbrain parenchyma. Intraoperatively and immediately postoperatively, thedecellularization zones appeared as hypoechoic foci with needle tractsappearing as distinct hyperechoic regions (FIG. 2).Neurosonographically, at the 24 hour examination the IREdecellularization zone was hypoechoic with a hyperechoic rim (FIG. 2).Compared to the 1 hour post-operative sonogram, the IRE decelluarizationzone appeared slightly larger (1-2 mm increase in maximal, twodimensional diameter). EEG performed in the post-operative periodrevealed focal slowing of the background rhythm over the right temporalregion in association with the decelluarization zones.

Macrolevel and histologic verification of treating cells: The brain wascollected within 2 hours of the time of death and removed from thecranium. Care was taken to inspect soft tissues and areas of closurecreated at the time of surgery. The brain was placed in 10% neutralbuffered formalin solution for a minimum of 48 hours. Then, the brainwas sectioned at 3 mm intervals across the short axis of the brain, inorder to preserve symmetry and to compare lesions. Following grossdissection of fixed tissues, photographs were taken of brain sections inorder to document the position and character of lesions, as shown inFIG. 3. Readily apparent in gross photographs of the sectioned brain arelesions created either by the physical penetration of brain substancewith electrodes or created by the application of pulse through theelectrodes. There are relatively well-demarcated zones of hemorrhage andmalacia at the sites of pulse delivery.

Microscopic lesions correlated well with macroscale appearance. Areas oftreatment are represented by foci of malacia and dissociation of whiteand grey matter. Small perivascular hemorrhages are present and there issparing of major blood vessels (see FIG. 4B). Notable in multiplesections is a relatively sharp line of demarcation (approximately 20-30micrometers) between areas of frank malacia and more normal, organizedbrain substance (see FIG. 4A).

Analysis to determine IRE threshold: To determine the electric fieldneeded to irreversibly electroporate tissue, one can correlate thelesion size that was observed in the ultrasound and MRI images with thatin the histopathological analysis to determine the percentage of lesiongrowth. Decellularized site volumes can be determined afteridentification and demarcation of IRE decellularization zones fromsurrounding brain tissue using hand-drawn regions of interest (ROI). Arepresentative source sample image is provided in FIG. 5.

Example 2 Use of IRE to Kill Brain Cells

There are advantages to a strategy to treat cancer using IRE. IRE totreat cancer has advantages over existing thermal ablation, includingthe ability to: monitor what area has been irreversibly electroporatedin real-time using ultra-sound or other imaging techniques; spare neuraltubes and blood vessels, enabling treatment in otherwise inoperableareas; preserve the microvasculature, which promotes rapid healing ofthe treated volume; predict the affected area using numerical modelingfor designing protocols; not be affected by blood flow as is thetemperature distribution during thermal therapies; image the success ofthe treatment using MRI or other imaging techniques; and administer theelectric fields in time periods of less than 5 minutes.

The present methods and devices provide a technology for treatment oftumors with IRE. Prior to the present invention, devices designed toirreversibly electroporate deep tissues did not exist. The experimentsconducted and reported in the prior art utilized reversibleelectroporation systems. These devices usually consist of large plateelectrodes (intended for transdermal drug delivery), needle electrodeswith a large probe (intended for targeting in or for small animalstudies), or cuvettes (used for in vitro applications). Applying anelectric pulse over the skin presents challenges for deep tissueapplications due to the significant voltage drop across the skin,generating considerable skin damage. (The same issue arises with anorgan containing an epithelial layer.) Other devices that use needleelectrodes are limited to superficial tumors. Furthermore, these toolshave a large mechanical housing making the treatment of subcutaneoustumors impossible without invasive surgery. A tool designed specificallyfor IRE for subcutaneous delivery of the electric field dramaticallyenhances the application space of IRE for tissue ablation.

To provide an initial proof of concept, a device according to theinvention was used to kill brain cells in vitro. Representing a uniquepathobiological phenomenon, high-grade canine gliomas exhibitessentially the same properties as human gliomas, including pathology(markers), genetics, behavior (invasiveness), lack of metastases, and asimilar clinical course of the disease. Dogs diagnosed with these tumorshave poor prognosis and most are humanely euthanized to prevent furthersuffering from the progression of the disease. Primary brain tumors(PBTs) account for 1-3% of all deaths in aged dogs where necropsy isperformed. The many similarities of glial tumors in people and dogs makethese tumors in dogs an excellent translational approach for newdiagnostic and treatment methods.

As shown in FIG. 6, cell proliferation of canine glioma cells wassignificantly reduced or eliminated by treatment with IRE. Morespecifically, FIGS. 1A-C show the results of J3T glioma cells aftertreatment with electric pulses of length 50 microseconds (μs) for 2electric fields (1000 V/cm and 1500 V/cm) and multiple numbers ofpulses. To develop the data shown in the figure, a WST-1 cellproliferation assay was performed on J3T glioma cells, and the datacollected 24 hours post-IRE treatment. Two electric fields (1000 and1500 V/cm) at 5 different pulse combinations were analyzed. A value ofrelative absorbance of 0.2 represents 100% cell death. Therefore, it isclear that for as low as 1000V/cm at 50 pulses total will achievecomplete cell death for 50 μs length pulses, proving this a viable IREtreatment parameter.

Example 3 Modeling of Electrode Shape and Placement

The present invention provides simple and elegant minimally invasivemicrosurgical tools to treat currently inoperable tumors in humans andanimals through IRE. Exemplary designs are shown in FIGS. 7A-E, 8A-C,9A-B, 10, and 11.

FIG. 7A depicts an example of a device 700 according to one embodimentof the invention. This embodiment is fully compatible with existingelectroporation electronics and comprises a surgical probe/electrode tip710 at its distal end, which includes both ground electrodes 711 andenergized electrodes 712. The device further comprises a universalconnector 750 at its proximal end. The device also comprises internalwiring 770 to deliver electrical impulses to the tip 710. The body ofthe device is defined by surface 718.

The size and shape of the IRE area is dictated by the voltage appliedand the electrode configuration and is readily predictable throughnumerical modeling. Therefore, different surgical tips can be fashionedto achieve the same therapeutic result. For example, tip 710 cancomprises retractable conductive spikes 713 emanating from a blunt endtip 710 and disposed, when deployed, at an acute angle to tip 710 (seeFIG. 7B). Alternatively, tip 710 can be fashioned as a point or needle,and can include retractable accordion-type conductive elements 714 (seeFIG. 7C). In other exemplary embodiments, tip 710 can comprise multipleretractable spikes 715 that, when deployed, emanate at 90° C. from tip710 (see FIG. 7D). Yet again, tip 710 can comprise retractableconductive spikes 716 emanating from a needle-end tip 710 and disposed,when deployed, at an acute angle to tip 710 (see FIG. 7E). FIGS. 7B, 7D,and 7E show probes with parallel circular channels 717 of approximately1 mm that protrude through the length of the electrode holder. Eachchannel has the capability of guiding individual 1 mm electrodes to thetreatment area. Towards the bottom of the holder, the channels deviatefrom their straight path at a specific angle. The electrodes can bePlatinum/Iridium with an insulating polyurethane jacket to ensurebiocompatibility, similar to materials that are used in DBS implants.Different protrusion depths of the electrodes within the tissue as wellas the applied voltage can be used to control the size of the treatedarea.

The devices can comprise interchangeable surgical tips, allowing forversatility in creating a device well suited for different tissues ordifferent sized or shaped tumors. Varying electrode diameters (varied inpart by selection of the type and length of deployable spikes) andseparation distances will be sufficient to ablate the majority of tumorsabout or smaller than 3 cm by selecting the appropriate voltages tomatch different tumor sizes and shapes. As shown in later figures, someof the embodiments of the device comprise an element at the tip tointroduce anti-cancer drugs for ECT, cytotoxic proteins, or otherbioactive agents into the targeted area.

While not depicted in detail, embodiments of the device comprise durablecarbon coatings over portions of the device that act both to insulatenormal tissue and to increase the efficiency of IRE pulsing.

With general reference to FIGS. 7A-D, in brain tumor IRE treatment, forexample, a single blunt-end device with embedded active and groundelectrodes can be used. In an embodiment not particularly depicted inthe figures, the device contains a primary blunt-end tip with a holedisposed in the end, for insertion through delicate, soft brain tissue.The device of these embodiments further comprises a secondary sharp tip,which can be deployed through the hole in the blunt-end primary tip,which allows for penetration into the tumor tissue, which can besubstantially more dense or hard, and not easily punctured by ablunt-end tip. In general, the device of the invention is typicallysimilar in dimensions (2 mm) to those already used in deep brainstimulation (DBS), which ensures that they are feasible for surgicalapplications. DBS uses electrodes in an FDA approved therapy toalleviate the symptoms of otherwise treatment-resistant disorders, suchas Parkinson's disease and dystonia. Furthermore, the electrodepositioning frame, which is used in stereotactic surgery in conjunctionwith imaging systems, can be used to position the surgical probes andensure that the position of the electrodes is optimal. Simulations of adesign similar to the one in FIG. 7A show treatment volumes comparableto typical brain tumors.

FIGS. 8A-C depict in more detail an embodiment of a tip 810 according tothe invention. FIG. 8A depicts an exploded view of tip 810, showingmultiple concentric layers of conducting 820 and non-conducting 830materials. An outer layer or sheath 860 of non-conducting material isshown with perforations 861. The outer, perforated layer 860 is disposedaround the concentric rings of materials, to allow for delivery ofbioactive substances to cells in proximity to the device when in use.Perforated layer 860 may be disposed in full, direct contact with theoutermost layer of the concentric ring structure, or may besubstantially separated from the ring structure by chamber 833 thatholds cooling fluid.

As shown in the cut-away depiction in FIG. 8A, device tip 810 hasmultiple alternating layers of conducting 820 and non-conducting 830materials surrounding an non-conducting inner core 831. In FIG. 8B, thetop and bottom conducting regions 820 are energized electrodes while themiddle conducting region 820 is a ground electrode. The presentinvention provides the conducting and non-conducting (insulative)regions in varying lengths to fine tune electrical field generation.More specifically, using imaging techniques directed at the tumor to betreated, a surgeon can determine what type of electrical field is bestsuited for the tumor size and shape. The device can comprise one or moremovable elements on the surface of the tip (not depicted) or can bedesigned such that one or more of the alternating conducting 820 ornon-conducting 830 elements is movable. Through movement and setting ofthe outer element(s) or inner elements 820 or 830, the surgeon canconfigure the device to deliver a three-dimensional electrical killingfield to suit the needs of the particular situation.

FIG. 8C depicts the concentric laminate structure of tip 810, viewedfrom the distal end along the distal-proximal axis, showing again thelaminate nature of the device.

In addition to changing charges, adapting the physical dimensions of theprobe also allows flexibility in tailoring the treatment area to matchthe dimensions of the tumor. By altering the electrode parameters,including diameter, length, separation distance, and type, it ispossible to conveniently tailor the treatment to affect only specific,targeted regions. In addition, developing an electrode capable ofaltering and adapting to these dimensional demands greatly enhances itsusability and adaptability to treatment region demands.

Example 4 Hollow Core Device

Many IRE treatments may involve coupled procedures, incorporatingseveral discrete aspects during the same treatment. One embodiment ofthe invention provides a device with a needle-like tip 910 with anincorporated hollow needle 990 with either an end outlet 991 (shown inFIG. 9A) or mixed dispersion regions 961 (shown in FIG. 9B). Such aconfiguration allows for highly accurate distribution of injectablesolutions, including those comprising bioactive agents. Use of such adevice limits the dose of treatment required as well as ensures thecorrect placement of the materials prior to, during, and/or after thetreatment. Some of the possible treatment enhancers that would benefitfrom this technology are: single or multi-walled carbon nanotubes(CNTs); chemotherapeutic agents; conductive gels to homogenize theelectric field; antibiotics; anti-inflammatories; anaesthetics; musclerelaxers; nerve relaxers; or any other substance of interest.

The schematics in FIGS. 9A-B show two basic hollow needle designs thatmay be implemented to enhance solution delivery prior to, during, orafter IRE treatment. They both have multiple conducting surfaces 920that may act as charged electrodes, grounded electrodes, or electricresistors, depending on the treatment protocol. FIG. 9A shows a hollowtip 910 for injection of agents at its end while FIG. 9B has distributedpores 961 throughout for a more generalized agent distribution. As shownin FIG. 9B, the pores are disposed in the non-conducting regions 930 ofthe device.

Example 5 Devices Comprising Active Cooling

In embodiments, the device comprises a cooling system within theelectrode to reduce the highly localized temperature changes that occurfrom Joule heating. During the electric pulses for IRE, the highestquantity of heat generation is at the electrode-tissue interface. Byactively cooling (for example, via water flow) the electrode during theprocedure, these effects are minimized. Further, cooling provides a heatsink for the nearby tissue, further reducing thermal effects. Thisallows more flexibility in treating larger tissue regions with IRE whilekeeping thermal effects negligible, providing a greater advantage forIRE over conventional thermal techniques. Cooling can be achieved byplacement of one or more hollow chambers within the body of the device.The cooling chambers can be closed or open. Open chambers can beattached at the proximal end to fluid pumping elements to allow forcirculation of the fluid (e.g., water) through the device during use.

Example 6 Movable Outer Sheath

In embodiments, the device comprises an outer protector that is designedto be movable up and down along the length of the device. FIG. 10depicts such a movable outer protector. More specifically, FIG. 10depicts a device 1000 comprising tip 1010 that includes outer protector1062 that can be moved up and down along the length of device 1000. Inpractice, outer protector 1062 is disposed fully or partially encasingouter sheath 1060. After or during insertion into tissue to be treated,outer protector 1062 is retracted partially to expose outer sheath 1060,which in the embodiment depicted comprises mixed dispersion outlets1061. As such, the number of dispersion outlets 1061 exposed to thetissue during treatment can be adjusted to deliver varying amounts ofbioactive agent to different portions of the tissue being treated. Anymechanism for movement of the outer sheath along the device may be used.In embodiments, screw threads are disposed on the upper portion of thedevice, allowing for easy adjustment by simple twisting of the outersheath. Alternatively, set screws may be disposed in the outer sheath,allowing for locking of the sheath in place after adjustment.

Example 7 System for IRE Treatment of Tumors

The invention provides a system for performing IRE tumor tissueablation. As depicted in FIG. 11, an exemplary system can comprise adevice 1100 reversibly attached to holder 1140. Holder 1140 can comprisetrigger 1141, which allows the user to control the flow of electricityfrom power source/controller 1142 to device 1100.

In this embodiment, device 1100 comprises further elements for use. Morespecifically, device 1100 comprises a height adjustment apparatus 1151at its proximal end to effect movement of outer sheath 1160. Outersheath 1160 further comprises markings or scores 1168 on its surface toindicate amount of movement of outer sheath 1160 after implantation ofdevice 1100 into tumor tissue.

Example 8 System for Controlling Multiple Electrodes

The invention provides a system for accurately controlling the distancesbetween multiple electrodes of singular or multiple polarities during acharge. The device places electrode types within an adjustable part of ahandle that may be maneuvered by a surgeon or attached to a harnesssystem, as described above. The adjustable portion of the handle may beused to control the relative depths of penetration as well as separationdistances of each electrode relative to one or more additionalelectrodes placed within the system.

Example 9 Modeling of Separation Distances Between Electrodes and HeatGeneration

The system and method of the invention can include the use of multipledevices for treatment of tumors. The devices can be implanted in thetumor at varying distances from each other to achieve desired cellkilling. Alternatively, the system and method can include the use of asingle device having multiple electrodes along its tip. Modeling ofplacement of multiple devices or a single device with multipleelectrodes in tissue was performed, and exemplary electrical fieldsgenerated are depicted in FIGS. 12A-E. The outputs depicted in thefigure demonstrate the variability in IRE treatment region that resultsfrom altering the separation distance of the conducting electrodesurfaces. More specifically, FIGS. 12A-C show three model outputs for2-dimensional needles (leftmost images) and an axis symmetric electrode(rightmost images). For all images, there were two charged surfaces, oneof 2500V and one of 0V. The distances between the electrodes were 0.5 cm(FIG. 12A), 1.0 cm (FIG. 12B), and 1.5 cm (FIG. 12C). From this data, itis clear that altering the distance leads to significantly differentelectric field distributions, and thus makes the distance an importantparameter to consider when developing IRE protocols for various tumorablation.

Numerical models representing two needles and an axis symmetric needleelectrode configuration have been developed to compare the increase intreatment area shown by the electric field distribution for the samethermal effects between 100 and 50 μs pulse lengths. The area/volume oftissue that increased by at least 1 degree Kelvin was determined for a100 μs pulse. This area/volume was then used for the 50 μs pulse todetermine the electric field magnitude that would cause the sameincrease in temperature. A contour line has been created within thesemodels to represent the region treated with the IRE threshold of700V/cm. The results are shown in FIG. 12D. More specifically, 2-Dneedle electrodes with 3.13 mm² area of tissue increased by one degreeKelvin for 100 μs pulse at 2500V/cm with 226.2 mm² area treated by IRE(FIG. 12D, left side) and 50 μs pulse at 3525V/cm with 325.6 mm² areaaffected by IRE (FIG. 12D, right side). Axis symmetric needle electrodewith 3.95 mm³ volume of tissue increased by 1 degree Kelvin for 100 μsat 1500V with 81.1 mm³ volume affected by IRE (FIG. 12E, left side) and50 μs pulse at 2120V with a 133 mm³ volume within IRE range (FIG. 12E,right side).

Example 10 Use of Different Tip Sizes

To provide exquisite control of electrical fields, and thus cellkilling, the size of the electrode tips may be adjusted. In addition toreal-time electrode manipulation capabilities, integrating multipleelectrode types within the same procedure can make a large impact onenhancing electric field distribution selectivity. This can be done byincorporating such variations as a needle electrode with a single probeor parallel needle electrodes with the conductive surface of one being adifferent dimension (e.g., longer) than the other. As shown in FIGS.13A-C, the electrical field output can be altered based on thearrangement of electrode types. More specifically, the figure showsmodel outputs displaying the electric field distribution for threeneedle electrodes, with a contour of 700V/cm. It can be seen that bymixing up the diameter of the electrodes (as displayed with each figure)within the same treatment, the shape and area of tissue treated by the700V/cm threshold can be manipulated greatly. FIG. 13A shows the use oftips having, from left to right, 2 mm diameter, 0.5 mm diameter, and 1mm diameter, providing a 700V/cm threshold of 215.41 mm². FIG. 13B showsthe use of tips having, from left to right, 1 mm diameter, 1 mmdiameter, and 0.5 mm diameter, providing a 700V/cm threshold of 243.26mm². FIG. 13C shows the use of tips having, from left to right, 1 mmdiameter, 2 mm diameter, and 0.5 mm diameter, providing a 700V/cmthreshold of 271.54 mm².

Example 11 Use of Multiple Electrode Charges

We have discovered that a highly customizable electric fielddistribution may be attained by combining multiple electrode chargeswithin the same pulse. This allows a highly customized and controllabletreatment protocol to match the dimensions of the target tissue. Inaddition, the invasiveness of the treatment may be decreased by reducingthe number of electrode placements required for treatment. In order todemonstrate the great flexibility in electric field distribution shape,2-dimensional and axis symmetric models were developed with 3 and 4electrode arrays along a single axis. The results are depicted in FIGS.14A-D. For development of the data, only the electric potentials of theelectrodes were manipulated to achieve the great flexibility needed inIRE treatment planning. For FIGS. 14A-B, four charged electrodes ofalternating polarity at 2500V and ground were used to develop a 2-Dreadout (FIG. 14A) and axis symmetric electrode configurations (FIG.14B). Four charged electrodes with the center two at 5000V and 0V andthe outer two electrodes at 2500V were used to develop a 2-D readout(FIG. 14C) and axis symmetric electrode configurations (FIG. 14D). Threecharged electrodes with the center one at 2500V and the outer two at 0Vwere used for 2-D (FIG. 14E) and axis symmetric electrode (FIG. 14F)configurations. Three charged electrodes with the center at 0V, leftelectrode at 5000V, and right electrode at 2500V for 2-D (FIG. 14G) andaxis symmetric (FIG. 14H) scenarios. Three charged electrodes with thecenter at 1750V, left electrode at 3000V and right electrode at 0V for2-D (FIG. 14I) and axis symmetric electrode (FIG. 14J) configurations.

Example 12 Thermal Effects for Long Duration Treatment

FIGS. 15A-D display the modeling outputs of thermal effects during atypical IRE treatment, but for extended treatment periods. The images inFIGS. 15A and 15C display the thermal effects without convectivecooling, while the images in FIGS. 15B and 15D have the same treatmentparameters, but incorporate convective cooling of the needle. FIGS. 15Aand 15B: IRE treatment with 3 needles (1 second post-IRE) without (FIG.15A) and with (FIG. 15B) convective cooling at the electrode-tissueinterface. It can be seen, particularly on the large center electrodethat the temperature of the tissue contacting the electrode is theregion of highest temperature without cooling, but is actually a lowertemperature than the peripheral regions of the tissue. FIGS. 15C and15D: IRE treatment with 3 needles (5 seconds post-IRE) without (FIG.15C) and with (FIG. 15D) convective cooling at the electrode-tissueinterface. It can be seen, particularly on the large center electrode,that the temperature of the tissue contacting the electrode is theregion of highest temperature without cooling, but is actually a lowertemperature than the peripheral regions of the tissue.

Example 13 Altering the Diameter and Shape of Electrodes

We have done some preliminary studies and determined that the electricfield distribution may be altered, and thus controlled, by changing thediameter and shape of the electrode between the conducting surfaces.This fact can be used to design and develop an electrode with anexpandable/contractible interior and deformable exterior to change itssize in real-time before or during a treatment to alter, and thusspecify the electric field distribution in a manner that may bedesirable during treatment. The ability to adjust this dimension inreal-time is made additionally useful by the fact that a significantlysmaller electrode may be inserted to keep it minimally invasive, andthen expand the dimension once the electrode has reached the targettissue. In embodiments, the invention includes the use of a balloonbetween regions of charge that may be inflated/deflated during treatmentto alter field distribution. FIGS. 16A-C depict modeling of a bulgingregion between the charges in a bipolar electrode. Three differentmodels that study the inclusion of a balloon between the two electrodesin a bipolar design are shown. FIG. 16A (861.21 mm³ treated area) has noballoon for comparison purposes. The middle design of FIG. 16B (795.71mm³ treated area) has an elongated balloon that is in close proximity tothe electrodes. The bottom design of FIG. 16C (846.79 mm³ treated area)has a smaller balloon that helps distribute the electric field.

Example 14 Alternating Polarity

With the application of electric potentials, electrical forces may driveions towards one electrode or the other. This may also lead toundesirable behavior such as electrolysis, separating water into itshydrogen and oxygen components, and leading to the formation of bubblesat the electrode-tissue interface. These effects are further exacerbatedfor multiple pulse applications. Such effects may cause interferencewith treatment by skewing electric field distributions and alteringtreatment outcomes in a relatively unpredictable manner. By altering thepolarity between the electrodes for each pulse, these effects can besignificantly reduced, enhancing treatment predictability, and thus,outcome. This alternating polarity may be a change in potentialdirection for each pulse, or occur within each pulse itself (switch eachelectrode's polarity for every pulse or go immediately from positive tonegative potential within the pulse at each electrode).

Example 15 Bipolar and Monopolar Electrodes

Using a bipolar electrode with 4 embedded electrodes, one can use themiddle two electrodes to inject a sinusoidal current (˜1-5 mA) that islow enough in magnitude to not generate electroporation and measure thevoltage drop across the remaining two electrodes. From this setup onecan calculate the impedance of the tissue and gather the conductivity ofthe tissue which is needed for treatment planning. One can do thisanalysis in a dynamic form after each electroporation pulse.Conductivity increases as a function of temperature and electroporation;therefore, for accurate treatment predictions and planning, the dynamicconductivity is needed and we can use the bipolar or unipolar electrodesto map the conductivity distribution before IRE treatment and during toadjust the pulse parameters.

Example 16 Parameters

The following are parameters that can be manipulated within the IREtreatments discussed herein.

Pulse length: 5 μs-1 ms

Number of pulses: 1-10,000 pulses

Electric Field Distribution: 50-5,000 V/cm

Frequency of Pulse Application: 0.001-100 Hz

Frequency of pulse signal: 0-100 MHz

Pulse shape: square, exponential decay, sawtooth, sinusoidal,alternating polarity

Positive, negative, and neutral electrode charge pulses (changingpolarity within probe)

Multiple sets of pulse parameters for a single treatment (changing anyof the above parameters within the same treatment to specialize outcome)

Electrode type

-   -   Parallel plate: 0.1 mm-10 cm diameter    -   Needle electrode(s): 0.001 mm-1 cm diameter    -   Single probe with embedded disk electrodes: 0.001 mm-1 cm        diameter    -   Spherical electrodes: 0.0001 mm-1 cm diameter

Needle diameter: 0.001 mm-1 cm

Electrode length (needle): 0.1 mm to 30 cm

Electrode separation: 0.1 mm to 5 cm

Example 17 Specific Conductivity

The methods used to model tissue ablation are similar to the onesdescribed by Edd and Davalos for predicting IRE areas based on theelectric field and temperature distribution in the tissue (Edd, J. F, etal., 2007, “Mathematical modeling of irreversible electroporation fortreatment planning.”, Technology in Cancer Research and Treatment.,6:275-286.) The methods are disclosed in Garcia et al., “Irreversibleelectroporation (IRE) to treat brain cancer.” ASME Summer BioengineeringConference, Marco Island, Fla., Jun. 25-29, 2008.

We have modeled a new electrode design for the application of IRE inbrain tissue. According to our results, IRE can be an effectivetechnique for minimally invasive brain tumor removal. The treatment doesnot induce substantial thermal effects in the brain, protecting theintegrity of this organ, which is susceptible to small fluctuations intemperature. In an embodiment of the method of the invention, the methodincludes delivering electrical signal(s) through tissue to determine itselectrical properties before administering IRE by monitoring the voltageand current. Following from that, one may apply intermittent andpost-IRE pulse(s), which can be used to determine the success of theprocedure and adjust IRE pulse parameters.

Specific conductivity can be important for treatment planning ofirreversible electroporation (IRE). For many applications, especiallywhen treating tumors in the brain, the volume (area) of IRE must bepredicted to maximize the ablation of the tumorous tissue whileminimizing the damage to surrounding healthy tissue. The specificelectrical conductivity of tissue during an irreversible electroporation(IRE) procedure allows the physicians to: determine the currentthreshold; minimize the electric current dose; decrease the Jouleheating; and reduce damage to surrounding healthy tissue. To measure thespecific conductivity of tissue prior to an IRE procedure the physicianmust: establish the electrode geometry (shape factor); determine thephysical dimensions of the tissue; apply a small excitation AC voltagesignal (1 to 10 mV); measure the AC current response; calculate thespecific conductivity (σ) using results from the prior steps. Thisprocedure will not generate tissue damage (low amplitude AC signals) andwill supply the physician (software) with the required information tooptimize IRE treatment planning, especially in sensitive organs like thebrain which is susceptible to high electrical currents and temperatures.Thus, the IRE procedure is well monitored and can also serve as afeedback system in between series of pulses and even after the treatmentto evaluate the area of ablation.

Special Cases for electrode geometry:

Nomenclature (units in brackets):

-   -   V_(e)=voltage on the hot electrode (the highest voltage), [V]    -   R₁=radius of electrode with highest voltage (inner radius), [m]    -   R₂=radius at which the outer electrodes are arranged (outer        radius), [m]    -   i=total current, [A]    -   L=length of cylindrical electrode, [m]    -   σ=electrical conductivity of tissue, [S/m]

Electrical conduction between a two-cylinder (needle) arrangement oflength L in an infinite medium (tissue). It is important to note thatthis formulation is most accurate when L>>R₁,R₂ and L>>w. The electricalconductivity can be calculated from,

$\sigma = \frac{i \cdot S}{V_{e}}$where the shape factor (S) corresponding to the electrode dimensions andconfiguration is given by,

$\frac{2 \cdot \pi \cdot L}{\cosh^{- 1}\left( \frac{{4 \cdot w^{2}} - \left( {2 \cdot R_{1}} \right)^{2} - \left( {2 \cdot R_{2}} \right)^{2}}{8 \cdot R_{1} \cdot R_{2}} \right)}$

The specific conductivity (σ) of the tissue can be calculated since thevoltage signal (V_(e)) and the current responses (i) are known.

Explanation of electrical concepts: By using the bipolar electrodedescribed in the priority document, one can apply a small excitation ACvoltage signal (1 to 10 mV),V(t)=V ₀ Sin(wt)where V(t) is the potential at time t, V₀ is the amplitude of theexcitation signal and w is the frequency in radians/s. The reason forusing a small excitation signal is to get a response that ispseudo-linear since in this manner we can determine the value for theimpedance indicating the ability of a system (tissue) to resist the flowof electrical current. The measured AC current (response) that isgenerated by the excitation signal is described byI(t)=I ₀ Sin(wt+q)where I(t) is the response signal, I₀ is the amplitude of the response(I₀ ¹V₀) and q is the phase shift of the signal. The impedance (Z) ofthe system (tissue) is described by,Z=(V(t))/(I(t))=(V ₀ Sin(wt))/(I ₀ Sin(wt+q))=Z ₀ Sin (wt))/(Sin(wt+q))It is important to note that the measurement of the response is at thesame excitation frequency as the AC voltage signal to preventinterfering signals that could compromise the results. The magnitude ofthe impedance |Z₀| is the electrical resistance of the tissue. Theelectrical resistivity (W m) can be determined from the resistance andthe physical dimensions of the tissue in addition to the electrodegeometry (shape factor). The reciprocal of the electrical resistivity isthe electrical conductivity (S/m). Therefore, after deriving theelectrical resistivity from the methods described above, theconductivity may be determined.

Example 18 Use of Nanoparticles in IRE

Despite its mechanism of action, a major disadvantage of IRE in terms ofcancer treatment is that the pulsing protocol cannot distinguish betweenhealthy cells and tumor cells. Additionally, the voltage that is appliedduring treatment is limited to the maximum voltage that can be deliveredto the tissue without inducing Joule heating. Joule heating can lead todisruption of the extracellular matrix, nerve damage, and coagulation ofthe macrovasculature, which are undesirable treatment outcomes. Thisinvention simultaneously addresses both of these deficiencies throughthe incorporation of particles, and preferably nanoparticles ormicroparticles of very small size, into pulsed electric field therapies.This Example provides data showing that the use of small microparticlesand nanoparticles in IRE can increase treatment area without the need toincrease the applied voltage, which would result in thermal damage.

The electric field to which a cell is exposed determines whether or notit will undergo IRE or supra-poration. The typical electric fieldthreshold, which varies as a function of the cell type and pulsingparameters (frequency, duration, and number), is roughly 600 V/cm forIRE. If nanoparticles raise the electric field above the threshold forIRE, they can be incorporated into pulsed electric field therapies toexpand the treatment area or to lower the necessary applied voltage toinduce IRE, which reduces the extent of thermal damage. Conducting,semi-conducting, and insulating nanoparticles can be used to enhance theelectric field. The permittivity of the nanoparticles is not asignificant contributing factor to the calculated electric fielddistribution when the frequency of the applied field is below 1 MHz. Formaterials with a conductivity ratio below 1, a sphere or rod-shapednanoparticle oriented perpendicular to the applied field should beemployed, and for materials with a conductivity ratio above 1, a sphereor rod-shaped nanoparticle oriented parallel to the applied field shouldbe employed. See, for example, FIG. 18.

An assessment of the treatment area enhancement following IRE with theinclusion of nanoparticles is shown in FIG. 21. The developedtwo-dimensional finite-element model represents two parallel needleelectrodes (1 mm in diameter separated by a distance of 2 cm) insertedwithin a tumor. It is assumed that the tumor was initially atphysiologic temperature (310.15 K), and the simulation was run for asingle, 50 μs pulse with an applied voltage of 1500 V set along theboundary of one of the electrodes with the other set as ground. Theresults indicate that if nanoparticles delivered to the tumor canamplify the electric field by a factor of 2 such that an area that waspreviously at 250 V/cm meets an electric field threshold of IRE (takenin this example to be 500 V/cm), then the treatable area will beincreased by a factor of 4. Further, the predicted temperature rise of 1K is far less than that required to induce thermal damage from thedenaturation of proteins. As mentioned, by expanding the treated areawithout increasing the voltage applied through the electrodes, we willbe able to treat infiltrative cancer cells beyond the tumor margin forpreventing tumor recurrence and metastasis without inducing thermaldamage.

FIG. 19 depicts a bar graph showing cell viability as a function ofelectric field for 99, 500 μs pulses delivered at a frequency of 0.5 Hzwith a voltage ranging from 0-100 V (across 2 mm gap electrode). Morespecifically, the experimental evidence shown in FIG. 19 shows theability of multi-walled carbon nanotubes to lower the electric fieldthreshold for IRE. Human metastatic breast cancer cells (MDA-MB-231)were treated in vitro and in suspension with and without the inclusionof nanotubes. Nanotubes (0.5 mg/ml) were suspended in DEP buffer (8.5%sucrose [weight/volume], 0.3% glucose [weight/volume], and 0.725% RPMI[volume/volume]) supplemented with Pluronic 108 NF (BASF) for uniformdispersion, and cells were resuspended directly in this solution.Following treatment, cell viability was assessed through a trypan bluedye exclusion assay. Trypan blue was used to stain cells with acompromised plasma membrane, while viable cells remained unstained.Cells were counted conventionally on a hemacytometer with two trials pertreatment group (n=2). The results indicate that multi-walled nanotubescaused enhanced cell death in an applied electric field of 500 V/cm,whereas cells treated with the same pulsing parameters without theinclusion of nanotubes remain significantly more viable.

Computational FEMs for predicting the transmembrane potential acrosscells placed in a uniform electric field indicate that the inclusion ofmicro- and nanospheres can have a significant impact in terms ofaltering the induced transmembrane potential (ITV). More specifically,the data in FIG. 20 show that, when polystyrene beads (1 μm diameter)are inserted around one half of a cell (14 μm diameter) in a 1000 V/cmuniform DC and 1 MHz AC electric field, an enhanced electrical field iscreated. When the applied pulses are DC, this electric field enhancementcan alter the ITV and make the cell more susceptible to IRE. However,when the frequency of the applied field is larger than the inverse ofthe relaxation time of the cell (typically around one microsecond), thetransmembrane potential is inversely proportional to the frequency.Therefore, at 1 MHz, the transmembrane potential does not reach valuesabove 1 V, which are required for IRE. The enhanced cell death ispresumably due to the localized electric field enhancement around themicrospheres. Because the microspheres are insulators, they maintaintheir charged dipole orientation even in high frequency fields. In thesesimulations, the cell membrane boundary is treated as a distributedimpedance, while the microsphere membranes are treated as continuous.

Example 19 Use of IRE and Nanoparticles with Modified Surface Chemistry

IRE has been shown to promote tumor regression. However, it cannotselectively kill cancer cells within a tumor mass without also killinghealthy cells. The selectivity of pulsed electric field therapies can beenhanced through the use of nanoparticles. The surface of nanoparticlescan be functionalized to target specific cancer cells with variousantibodies and chemical compounds. Due to the ability of certainnanoparticles to enhance the electric field, and the ability offunctionalized nanoparticles to target cancer cells, electric pulseprotocols can be optimized such that only cancer cells with selectivelybound nanoparticles experience a localized electric field above thethreshold for achieving IRE, and healthy cells remain intact. Theconcept of use of functionalized surfaces for IRE is provided in FIG.17, in the context of functionalizing an electrode tip. The same conceptand general chemistry can be used to functionalize nanoparticles tocreate a specific association of the nanoparticles with target cells.This methodology can be employed to purge the body of cancer cellswithin and beyond the treatment margin, while maintaining proper organfunction. Tumors can be up to 80% healthy cells, and selectivelydestroying the cancer cells, including cancer stem cells (CSCs), reducesthe potential for tumor recurrence.

Example 20 Use of IRE with Nanoparticles Incorporating Drugs for CancerTreatment

A portion of the treatment area that does not experience an electricfield above the threshold for IRE still undergoes reversibleelectroporation. Therefore, microspheres and nanospheres can be used ascarriers to get drugs, such as chemotherapeutic agents, into cellsthrough reversible electroporation. Under normal conditions, these drugswould not be able to permeate the plasma membrane. Additionally, thepulsing parameters can be tuned to electrophoretically drive themicrospheres or nanospheres loaded with drugs through the reversiblepores. This addition to conventional IRE therapy can help to furtherreduce tumor recurrence.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

The invention claimed is:
 1. A method of treating a subject sufferingfrom a neoplasia, said method comprising: identifying a treatment areain the subject, wherein the treatment area comprises neoplasia;administering a first therapeutically effective amount of nanoparticlesto the subject in need thereof such that at least some of thenanoparticles come in close proximity to neoplastic cells of theneoplasia; positioning at least one electrode into or adjacent theneoplastic cells; applying an electric field to the treatment area bydelivering electrical pulses from the at least one electrode to causenon-thermal irreversible electroporation of the neoplastic cells; andorienting the nanoparticles in relationship to the electric field suchthat the electric field is enhanced, the treatment area is enlarged, amore precisely defined treatment margin is achieved, or tumor marginsare more selectively treated.
 2. The method of claim 1, wherein the stepof identifying the treatment area further comprises identifying aneoplasia presented as any one of: a leukemia, a non-solid tumor, asolid tumor, and tumors in the brain, bone marrow, liver, prostate,kidney, breast, and pancreas.
 3. The method of claim 1, wherein thesubject is a human.
 4. The method of claim 1, wherein the electricalpulses are each 50 microseconds or less.
 5. The method of claim 1,wherein the step of positioning further comprises positioning two ormore electrodes.
 6. The method of claim 1, wherein the step ofdelivering electrical pulses further comprises limiting the total chargedelivered to the subject to minimize disruption of or damage to healthytissue surrounding the neoplastic cells.
 7. The method of claim 1,wherein the method comprises administering 50 pulses or more.
 8. Themethod of claim 1, wherein the method further comprises monitoringcurrent delivered by the electrodes in real-time and, based on thatmonitoring, preventing excessive charge delivery to healthy tissue. 9.The method of claim 1, wherein the method further comprises amplifyingthe applied electric field around the nanoparticles such that theelectric field threshold required for inducing irreversibleelectroporation of the treatment area is lowered, and the treatment areais enlarged.
 10. The method of claim 1, wherein the method furthercomprises delivering pulses of 100 microseconds or less.
 11. The methodof claim 1, wherein the method further comprises delivering electricalpulses at a voltage gradient of about 500 V/cm to about 1500 V/cm. 12.The method of claim 1, wherein the step of administering nanoparticlescomprises administering microparticles.
 13. The method of claim 12,wherein the nanoparticles range in size from about 1 nm to about 100,000nm.
 14. The method of claim 12, wherein the step of administeringnanoparticles further comprises administering nanoparticles selectedfrom the group comprising spherical nanoparticles, rod-shapednanoparticles, fullerenes, endohedral metallofullerenes (EMFs),trimetallic nitride template endohedral metallofullerenes (TNT EMFs),single-walled and multi-walled carbon nanotubes, gold nanorods, silvernanorods, single-walled and multi-walled boron/nitrade nanotubes, carbonnanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes,nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles,nanorods, polystyrene beads, glass and polymer micro- and nano-spheres,biodegradable micro- and nano-spheres, cellulose nanocrystals, glassnanospheres, polystyrene particles, polymer nanospheres, goldnanoparticles, silver nanoparticles, carbon nanoparticles, and ironnanoparticles, conducting nanoparticles, semi-conducting nanoparticles,and insulating nanoparticles, or a combination of two or more of these.15. The method of claim 12, wherein the step of administeringnanoparticles comprises administering nanoparticles comprising amodified surface chemistry to cause nanoparticles to be in closeproximity to cell membranes and cause localized destruction of thetreatment area, while leaving untargeted cells intact.
 16. The method ofclaim 12, wherein the step of administering the nanoparticles furthercomprises selectively binding the nanoparticles to the neoplastic cells,such that the electric field of the neoplastic cells is above thethreshold required for inducing irreversible electroporation of theneoplastic cells.
 17. The method of claim 12, wherein the step ofadministering the nanoparticles further comprises administering thenanoparticles outside of the treatment area.
 18. The method of claim 12,wherein the nanoparticles comprise one or more targeting moietiesselected from proteins, antibodies and their fragments, peptides,nucleic acids, aptamers, small molecules, vitamins and carbohydrates.19. The method of claim 12, wherein the nanoparticles have aconcentration in solution ranging from 0.1 μg/ml to 100 mg/ml.
 20. Themethod of claim 1, wherein the method further comprises orienting thenanoparticles such that the nanoparticles are parallel or perpendicularto the applied electric field.
 21. The method of claim 1, wherein duringthe step of administering the nanoparticles, the method furthercomprises systemically administering the nanoparticles with targetingantibodies, wherein the targeting antibodies are capable of binding tothe neoplastic cells.
 22. The method of claim 1, wherein the methodfurther comprises cooling the at least one electrode.
 23. The method ofclaim 1, wherein the method further comprises reversibly electroporatingtissue surrounding the treatment area and introducing at least onebioactive agent into the surrounding tissue using the nanoparticles. 24.The method of claim 1, wherein the step of administering the firsttherapeutically effective amount of nanoparticles further comprisesadministering the nanoparticles after the step of delivering theelectrical pulses to the treatment area.
 25. The method of claim 1,wherein the method further comprises administering a secondtherapeutically effective amount of the nanoparticles after the step ofdelivering electrical pulses to the treatment area.
 26. A method oftreating a subject suffering from a neoplasia, said method comprising:identifying a treatment area in the subject, wherein the treatment areacomprises neoplasia; administering a therapeutically effective amount ofnanoparticles to the subject in need thereof such that at least some ofthe nanoparticles come in close proximity to neoplastic cells of theneoplasia; positioning at least one electrode into or adjacent theneoplastic cells; applying an electric field to the treatment area bydelivering electrical pulses from the at least one electrode to causeelectroporation of the neoplastic cells; and orienting the nanoparticlessuch that the electric field is enhanced, the treatment area isenlarged, a more precisely defined treatment margin is achieved, or thetumor margins are more selectively treated.
 27. The method of claim 26,wherein the step(s) of administering the nanoparticles comprisesadministering microparticles.
 28. The method of claim 26, wherein theadministering of the nanoparticles in a manner that enlarges thetreatment area or more selectively treats tumor margins comprises one ormore of allowing proximity of the nanoparticles to a cell membrane ofthe neoplastic cells through targeting moieties, aligning thenanoparticles relative to the cell membrane, infusion of thenanoparticles to the neoplasia or bloodstream, and relying on anenhanced permeability and retention effect.